RESUMEN
The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Virus de la Leucemia Murina de Moloney , ADN Polimerasa Dirigida por ARN , Streptococcus pyogenes , Animales , Humanos , Ratones , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Virus de la Leucemia Murina de Moloney/genética , ADN Polimerasa Dirigida por ARN/genética , Streptococcus pyogenes/genética , Desoxirribonucleasa I/genéticaRESUMEN
Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.